A high-performance liquid chromatographic method is described which is suitable for the determination of the common antiseptic chlorhexidine in urine. The method employs Sep-Pak cartridges to remove chlorhexidine from the urine matrix. Chromatographic separation was achieved on a C18 reversed-phase column using a mobile phase of methanol-20 mM sodium acetate solution (60:40) adjusted to pH 5 with glacial acetic acid. An ion-pair agent (pentadecafluorooctanoic acid) was used at a concentration of 100 micrograms ml-1 X 3-Bromobenzophenone was used as chromatographic standard (kappa' = 4.0). 4-Bromobenzophenone (kappa' = 3.9) or dibenzal hydrazine (kappa' = 4.4) may also be used. A series of urine samples was analysed and no interferences were observed. The method is simple and rapid with a total analysis time of ca. 30 min.